The use of monoclonal antibodies against connective tissue proteins to establish the collagen profile of histological, cytological and biological fluid samples is an advantageous approach to disease diagnosis and therapy monitoring. Because of the high specificity and sensitivity of monoclonal antibodies, early detection of certain collagen-related pathological conditions is possible as is early assessment of the efficacy of certain therapeutic programs.
The fusion of mouse myeloma cells to spleen cells from immunized mice by Kohler and Milstein in 1975 [Nature 256:495 (1975)] demonstrated for the first time that it was possible to obtain continuous cell lines making homogeneous (so-called "monoclonal") antibodies. Since this nominal work, much effort has been directed toward the production of various hybrid cell lines (also called "hybridomas") and to the use of the antibodies made by these hybridomas for various scientific investigations. While the general technique for the preparation of hybridomas and monoclonal antibodies is well known, there are many difficulties met and variations required for each specific case. There is no assurance, prior to attempting to prepare a given hybridoma, that the desired hybridoma will be obtained, that it will produce antibody if obtained, or that the antibody so produced will have the desired specificity. In fact polyreactive monoclonal antibodies are a major obstacle in the generation of specific monoclonal antibodies to collagen Type II. Chichester et al. J. Immunol. Methods 140:259 (1991).
Immunoassays utilizing polyclonal antibodies have been previously described for the measurement of collagen types I, II and III [Henrotin et al. J. Immunoassay 11(4):555 (1990); Gosslau et al. J. Immunol. Methods 29:71 (1979); Rennard et al., Anal. Biochem. 104:205 (1980); and Bellon, Anal. Biochem. 150:188 (1985)], and propeptides of collagen types I and III [Parfitt et al. J. Bone Mineral Res., 2:427 (1987); and Petersen et al. Arth. Rheum. 29:592 (1986)]. Potential disadvantages in employing polyclonal antibodies for developing immunoassays are their heterogeneous composition and variation in the consistency of sera from animal to animal.
Monoclonal antibodies are ideal for employment in quantitative assays because they are epitope defined and can detect minor structural differences between individual types of collagen. However, the testing for monospecificity of monoclonal antibodies needs to be extensive. Once characterized, monoclonal antibodies can be produced with uniform consistency in large quantities for use in immunoassays and do not require serial affinity chromatography purification.
Chondrocyte cultures are routinely employed for the screening of antiarthritic agents. In primary chondrocyte culture systems, collagen other than Type II can be present in relatively high levels. Chondrocytes in culture tend to change phenotype over time switching from Type II collagen to Types I and III producing cells. In primary chondrocyte cultures, fibroblast contamination is also a common occurrence. Fibroblasts produce collagen Types I, III, IV, V and VI which can interfere with the measurement of Type II collagen. Thus, a sensitive method is required to detect different types of collagen (or degradative peptides thereof) in chondrocyte cultures.
The identification and quantitation of collagen Type II derived peptides in serum, lavage fluids of joints, or synovial fluids of animal models with experimental arthritis or humans with arthritis will provide new and important information on pathogenesis of the disease (e.g., which enzymes are involved in the degradation process), and on the severity of the degradation of cartilage at that time point. The collagen peptide concentration in serum will provide information on the effectiveness of drug treatment on cartilage collagen breakdown and collagen peptide release into body fluids. The ease of taking blood samples from patients or experimental animals will allow a close monitoring of the cartilage destruction.